An Introductory Guide to Light Microscopy

16 Apr to 14 May 2007

Michael Hooker Microscopy Facility

Michael Chua Wendy Salmon [email protected] [email protected] 843-3268 966-7051 6007 Thurston Bowles 6129 Thurston Bowles

An Introductory Guide to Light Microscopy - Five Talk Plan

• Apr 16. A brief perspective of microscopy, theory of operation, key parts of a typical microscope for transmitted light, Kohler illumination, the condenser, objectives, Nomarski, phase contrast, resolution

• Apr 23. Fluorescence: Why use it, fluorescence principals, contrast, resolution, filters, dichroic filter cubes, immuno staining, fluorescent proteins, dyes.

• Apr 30. Detectors, sampling & digital images: Solid state digital cameras, Photomultipliers, noise, image acquisition, Nyquist criterion/resolution, pixel depth, digital image types/color/compression

• May 07. Confocal Microscopy: Theory, sensitivity, pinhole, filters, 3-D projection/volume renders

• May 14. Advanced Fluorescence/Confocal: Live cell imaging, co- localization, bleed through/cross talk, FRAP, fluorescence recovery after photobleaching, deconvolution Today’s Plan • Brief perspective / evolution of imaging • Basic Optics • Parts of a microscope & Kohler Illumination • Objectives • Modes of Imaging common in Biomedical Research History and Evolution

• 4 Centuries of light microscopy!

Zeiss compound microscope with confocal scan head (2001)

~400 years Janssen

Zacharias Janssen was a Dutch spectacle-maker credited with inventing the first compound microscope in ~1590. Magnifications ~3x to ~9x. Huygens Christiaan Huygens (1629–1695) was a Dutch mathematician, astronomer and physicist developed an improved two lens eye piece. Optical errors in two half curved lenses tend to cancel out. Can get more magnification.

Hooke Robert Hooke improved the design of the new compound microscope, including a light source ~1655. Developed the micrometer. Resolution ~5 um. Leeuwenhoek

Anton van Leeuwenhoek simple microscope (~1675) used a single lens which yielded high magnifications (~70x to ~300x) and excellent resolution (~1 um). He reported seeing many kinds of microorganisms including bacteria! Chester Moore Hall & John Dollond

• Achromatic refracting lens was invented in 1733 by an English barrister named Chester Moore Hall • Patented by John Dollond.

Lister Tight focus

Spherical aberration

Joseph Jackson Lister (1786-1869) design and construct superior complex lenses by combining lenses of crown and flint glasses of different dispersion, but separated in order to both correct chromatic aberration and minimize spherical aberration. Abbe Ernst Abbe (1840-1905) applied mathematical principles to the design of lenses, which dramatically facilitated the manufacturing high quality optical instruments by the Carl Zeiss corporation. Abbe's realization that the performance of a microscope was limited by the diffraction of light was not well accepted for decades.

The Abbe limit formula Köhler In 1893 August Köhler (1866-1948) invented a method of providing optimum illumination of a microscope specimen while working at the Zeiss Corporation. Improved resolution and evenness of light illumination made photomicrography possible. Zernike Frederik Zernike (1888–1966) invented phase contrast microscopy in 1933, a way to see unstained cells. Nobel prize 1953.

Nomarski Georges Nomarski (1919-1997) developed the differential interference contrast (DIC) microscopy technique, which bares his name. Ploem

Johas Ploem invented the epi-illumination cube used in fluorescence microscopy. Dichroic filter cube

Arc Lamp (white light) Sample Excitation

Filter Emission Filter

Dichroic Splitter Fluorescence Objective image Inoue & D. Allen & N. Allen Video enhanced microscopy – use electronic camera and computer generated contrast enhancement.

Shinya Inoue Bob & Nina Allen Marvin Minsky 1957 Patented the Confocal Scanning Microscope: U.S.Patent 3013467

Practical Confocal microscope systems became available in the late 1980s. Yields improved contrast, resolution and optical sectioning. Sedat, Amos & Agard

1980s Digital deconvolution microscopy removes haze.

Mathematical Transformation + time

+ a powerful computer with extensive storage History - Recent Evolution+ Dyes – fixed, vital, indicators Better resolution Immunostaining /Antibodies More sensitivity Molecular Biology Lower noise Illumination Faster detection Lasers Greater specificity Electronics Easier analysis Cameras – CCDs, Intensifiers, high speed Bigger storage Optics – AOTF, AOM, guide fibers New capabilities Computers Increased complexity Algorithms & software Increased cost Techniques – Time lapse, FRAP, FRET, FLIM More raw data Control systems – focus, x-y movement, shutters Live cell environmental control Optics – Light is an Electromagnetic Wave

•Wavelength (can see) •Intensity (can see) •Phase (can not see) •Polarization (can not see) c = νλ ν = frequency λ = wavelength c = 3x108 m/s velocity in a vacuum e.g. Green: 550 nm, 54x1012 Hz (c.f. cell phone 1.9 GHz, 16 cm) Optics - Reflection

• Angle of reflection equals angle of

incidence, θ1 = θ2 • No change in frequency or wavelength • At larger angles, polarization reflection favors polarization parallel to the mirror θ1 plane, since light is wave phenomenon normal θ2 • Can be due to EM wave interaction with: 1. Dielectric and conductor, e.g. silver coating on back of glass, 2. Plasmon – bouncing of EM waves off sea of vibrating electrons on metal surface, 3. Interface at materials of different refractive index. mirror E.g. Sunset light reflected off surface of the ocean Optics – Refraction 1

n1 n2

θ1 interface normal θ θr 2

• Light bent due to change in phase velocity in different media • No change in wavelength • Also get reflected component with larger difference in refractive indices

• Snell’s law: n1sin(θ1) = n2sin(θ2) • Wavelength dependent – more refraction with shorter wavelength

• Refractive index: n = c / velocitymedium , (c = velocityvacuum) Optics – Refraction 2 Lens

source image

n1 n2 c1 c2

Medium refractive index n1 < n2 Velocity in medium c2

When light arrives out of phase, there is destructive interference Optics - Diffraction

Pin hole

• Interaction of EM waves with small objects and edges – Interference effects – Summation/cancellation of in phase and out of phase waves • No change in frequency or wavelength • Will see later that diffraction limits optical resolution Optics – Interference examples λ/2 dielectric Interference filter

In phase -constructive interference

Out of phase - destructive In phase - interference constructive interference

metallic layer

Reflected component Destructive interference Coherent light

Out of phase - destructive λ interference

Slit interference Key Parts of a typical microscope

Note that the lamp is missing. Simplified Optical Path of a Compound Microscope

(Observation light path only)

Focal Image Plane Plane Virtual Image Image Real

Objective Tube Eye Lens Piece

Specimen Simplified Optical Path

Illumination and observation light paths

Focal Image Plane Plane

Eye Lamp Condenser Objective Tube Lens Piece

Problem: See image of lamp filament Kohler Illumination

Condenser has double apertures

1. Condenser aperture changes the cone angle of light at the specimen

2. Field diaphragm changes diameter of light at the specimen The Condenser

• …is your friend – When trying to find your sample • Minimum aperture gives: – Thick depth of focus (easy to find sample since greater better chance of being in focus) – High contrast (can see edges of colorless cells, and also see dust & scratches which is great for finding the sample) – Less light (but have more than enough anyway) – Poorer resolution (who cares when just locating sample) – When taking images • Maximum aperture provides: – Thinner depth of focus (less overlying material seen) – Lower contrast (more even background, no dust and scratches) – More light (good for dark samples) – Best resolution (camera will notice the difference but eye will not) Parts of a typical microscope MICROSCOPE COMPONENTS

Camera

Binocular Camera Adapter Eyepiece Epi-Condenser Epi-Lamp Housing Epi-Field Diaphragm Diaphragm Mirror: & Centering Filters Shutter Beam Switch Focus and Centering Magnification Changer Filter Cube Changer Slot for Analyzer Body Tube Focus, Centering Slot for DIC Prism Objective Nosepiece Objective Stage Trans-Lamp Housing Condenser: Diaphragm&Turret Mirror: Centering Focus and Focus Centering Slot for Polarizer Field Diaphragm

Upright Microscope Coarse/Fine Filters Lamp: Focus, Centering Stand Specimen Focus and Diffuser From E.D. Salmon Parts of a typical microscope Objectives The objective is the 1ST imaging element Determines the performance of the optical system

• Magnification • Numerical Aperture (NA) • Immersion: oil – glycerol – water – air 40x NeoPlan 0.4 NA cover slip thickness adjustment correction • Tube length system (infinity) • Cover slip (0.17 mm or 0 or variable) • WD = working distance • Corr = Correction Collar cover slip – iris – immersion medium • Field curvature and aberration correction: Plan Apo – Fluor – Acromat … Objectives - Magnification

• Note: more magnification (M) gives less light intensity at detector Due to inverse square law Often, lens elements that are also more absorbing • Brightness proportional to 1 / M2 • Color coded band shows magnification (roughly)

Brightness I α 1 / M2

4 units magnified 16 units

2x 1-1.5x black 2-2.5x brown 4x 4-5x red 10x yellow 20x green 40-50x light blue original 60-63x dark blue 1 unit 1 unit 100x white Objectives - Numerical Aperture (NA)

NA = n·sin(a) From Molecular Expressions a = half angle of cone of illumination n = refractive index of medium • Numerical aperture is related to light gathering and resolving power. • High NA leads to smaller working distance. • Transmission brightness is proportional to NA2 Numerical Aperture (NA) - resolution

NA = n·sin (a) , a = half cone angle objective n = refractive index maximum of medium imaging λ = wavelength cone angle

Wide Field d = λ / (2 NA) (d=distance) = 0.22 μm

Confocal rairy = 0.61 λ / NA / √2 a = 0.15 μm

2 Confocal raxial-fluor = 1.77 λ / NA = 0.4 μm Objectives - Immersion Refractive Indexes: • Dry (air) = 1.0003 • Water = 1.33 • Glycerol = 1.47 objective • Immersion oil = 1.52 air • Glass = 1.52 cover slip Snell’s law of refraction objective n1·sin(o1) = n2·sin(o2) oil cover slip n o1 2 air

From Molecular Expressions n1 glass o2

(n = refractive index) Objectives - Cover Slips

• For non-oil immersion objectives, the coverslip is really a lens element. • Use (number) #1.5 for many objectives • With higher magnification, non- immersion lenses with a cover slip correction collar, it is very important to adjust it appropriately

This objective’s correction collar lists cover slip thickness in mm. Set to 0.17 for #1.5 cover slips, and/or use maximum contrast method if your sample has structures with good contrast for empirical adjustment while viewing. From Molecular Expressions Objectives - Tube length • Modern scopes are “infinity” corrected – Allows focus by moving objective without change of magnification – Allows filters, cubes and other components between the objective and tube lens without causing distortion • Older scopes may be 160 mm • Need to match with microscope Objectives – Corrections

• Coverslip – 0 to 2 mm – Note: culture dishes are 1 mm thick • Immersion medium – oil, glycerol, water • Aperture – Max. NA - reduced NA – Used for matching darkfield condenser NA or reducing glare; but loose resolution and brightness Objectives – Special Properties

• DIC (Nomarki) • Phase Contrast

• WD = Working distance Objectives - Typical

Objective Type Spherical Aberration Chromatic Aberration Field Curvature correction Achromat 1 Color 2 Colors No Plan Achromat 1 Color 2 Colors Yes Fluorite 2-3 Colors 2-3 Colors No Plan Fluorite 3-4 Colors 2-4 Colors Yes Plan Apochromat 3-4 Colors 4-5 Colors Yes Care of Objectives

Gentle, gentle, gentle, gentle! When rotating objective turret: Focus away 1st. Slowly! When changing slides: Focus away. Remove slide slowly! When unscrewing objectives: Two hands! Don’t let drop. Store immediately in objective holder tube. Don’t leave on table.

Cleaning: • Should be infrequent with dry lenses. • Lens tissue or surgical cotton only! Never Kim Wipes, Kleenex, Q-Tips, etc. • With immersion lenses, blot front lens gently. Do not wipe or rub. • Dry or immersion lenses clean off residue or oil use coated lens cleaner (pH ~6.0 to 7.0, non-ionic detergent, short chain alcohol) Objective & Condenser NA – Resolution

objective NAobj = no·sin (a) , a = half cone angle cone angle no = refractive index of objective side medium λ = wavelength

NA = n ·sin (c) n = refractive index cond c c a n of condenser imm. medium o c Approx. resolution d = λ / 2 NA nc

Resolution with condenser

d = λ / (NAobj + NAcond ) Condenser cone angle Common Modes of Imaging

• Transmitted – Wide field (standard transmitted) – Phase contrast – Nomarski (DIC) – Polarization (material science) • Epi-illumination (including confocal) – Fluorescence – Reflection (material science) Transmitted light – (wide field)

Live Buccal Epithelial cell (unstained) Wave Nature of Image Formation objective NA = n·sin (α) , α = half cone angle maximum imaging cone angle

α n

Ernst Abbe (1872) Image formation:- Collection of diffracted rays around sample by objective Interference of these rays in the image plane d = λ / (2n sin(α)) n = refractive index of medium λ = wavelength of light Phase Contrast

From Molecular Expressions Phase contrast Easy to set up. Works through plastic Loose resolution Normarski (Differential Interference Contrast, DIC)

(Polarizer) Birefringence Setup, setup, setup! 1. Kohler illumination 2. Cross polarizers 3. Push in Wollaston prisms 4. Adjust shear (Wollaston II) No birefringent material in light path, e.g. plastic, collagen

But easy to do once practiced. Nomarski enhances local gradients of refractive differences

Wide field Nomarski (DIC) Standard Light Microscope

• Transmitted light microscopy • Sample needs to be mostly transparent • Dyes give contrast/color

• Exciting light adds background – reduces contrast Fluorescence – Live Buccal Epithelial cells More next Monday

Transmitted Fluorescence FM 1-43 membrane dye Barely scratched the surface of light microscopy

• Some References – Fundamentals of Light Microscopy and Electronic Imaging, D. Murphy (2001) – Keller, E. H. 1995. Objective lenses for confocal microscopy. In Handbook of Biological Confocal Microscopy. pp. 111- 126. [Ed. Pawley, J. H.] Plenum press, New York – Microscopy from the Very Beginning, 2nd ed., Carl Zeiss Microscopy – Salmon, E. D. and J. C. Canman. 1998. Proper Alignment and Adjustment of the Light Microscope. Current Protocols in Cell Biology 4.1.1-4.1.26, John Wiley and Sons, N.Y. An Introductory Guide to Light Microscopy - Five Talk Plan

• Apr 23. Fluorescence: Why use it, fluorescence principals, contrast, resolution, filters, dichroic filter cubes, immuno staining, fluorescent proteins, dyes.

• May 07. Confocal Microscopy: Theory, sensitivity, pinhole, filters, 3-D projection/volume renders

• May 14. Advanced Fluorescence/Confocal: Live cell imaging, co- localization, bleed through/cross talk, FRAP, fluorescence recovery after photobleaching, deconvolution